| Budget Amount *help |
¥3,100,000 (Direct Cost: ¥3,100,000)
Fiscal Year 1998: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 1997: ¥2,100,000 (Direct Cost: ¥2,100,000)
|
|---|
| Research Abstract |
Heat-stable enterotoxin receptor (STaR/GC-C) is a member of membrane-associated guanylyl cyclase (GC) family. As compared with other GC receptors, following GC catalytic domain, STaR has prolonged carboxy-terminal tail which is about 60 amino acids longer than NPR-A (CC-A) and NPR-B (CC-B), and about 30 amino acids longer than GC-D, retGCl (GC-E), and retGC2 (GC-F). To elucidate the functional role of the additional carboxy-terminal tail, we examined the GC activities of two truncate STaR mutants, CDELTA1015 and CDELTA1023, which lack Gu^1^0^1^5-Phe^1^0^5^0 and Lys^1^0^2^3-Phe^1^0^5^0, respectively. After incubation with I jiM STa, the cells expressing CDELTA1O15 and CDELTA1023 accumulated about 20- and 10-folds higher cGMP than the cells expressing wild-typc STaR.The basal level of cGMP contents were not different between the cells with wild-type STaR and STaR mutants. Furthermore, the difference of CC activity was not observed at protein expression level. In addition, removal of carboxy-terminal tail of STaR induced not only high maximum level of cGMP-production but also high potential level of cGMP-produciion. These results suggest that the carboxy-terminal region of STaR might have an inhibitory function of STa-mediated GC activation, and therefore the lack of the carboxy-terminal region allowed to be activated high GC activity by STa.
STaR and its N-terminal extraceltular domain were prepared at a high level of expression from a system constructed of Sf21 insect cells and recombinant baculovirus. The recombinant STaR retained its binding activity to STa with a Rd value of 6.2x10^-^1^0^0M and cyclase catalytic activity at a level similar to those of STaR expressed jn mammalian cell lines, such as COS-7.
|
